Fecal pollution of public waterways, rivers, and lakes is a global problem of increasing concern, however, little is known about the survival and persistence of biological contaminants in the surrounding terrestrial environments including recreational beaches. In order to validate use of Escherichia coli (E. coli) as an indicator organism for possible health hazard exposure to fecal contamination, it is important to understand the physiology of survival and persistence of this organism and how it may correlate to pollution. This proposal encompasses three major aims. Aim 1 examines the role of abiotic and biotic factors in E. coli survival in the environment. The E. coli burden at three Wisconsin public beaches will be assessed during the late spring, summer, and early fall seasons of a 2-year time period. Point sources for coliform inputs will be characterized for each site and temperature, sand humidity, pO2, will be monitored throughout sampling. Total resident microbiota will be determined through microscopy to explore possible correlations between biofilms and/or microbial community interactions and E. coli survival in sand. Aim 2 examines each condition for survival using replicate sand plot models exposed to the same environmental conditions as nearby beachfronts by placement on an environmental green roof and inoculation with an E. coli strain originally isolated from a contaminated sand environment. Survival outcomes of the E. coli will be determined under individual and combined conditions of temperature stress, desiccation stress, and oxygen depravation, and in the presence or absence of resident microbiota. Aim 3 will focus on mechanistic studies to investigate the modulation of stress-related pathways, under control of global regulators such as alternative RNA polymerase sigma factors RpoS and RpoH. Levels of expression for RpoS and heat shock proteins (hsp) DnaK and GroEL will be studied in mid-log and stationary phase cells inoculated into sand plots. Mechanisms for survival under desiccation stress will be examined by assessing the role of trehalose synthesis as an osmoprotectant. These studies are unique in that protective responses are being studies in the sand environment, rather than under laboratory conditions. This collaboration allows for mechanistic studies of E. coli cell responses under environmental stressors while addressing an important public health issue. Such data will allow public health officials to formulate new strategies for the use of E. coli as a valid indicator of potential health risk and means for abatement of coliform pollution in recreational waterfronts. Importantly, the proposed work brings together investigators from two AREA grant eligible institutions to maximize the research experience for the undergraduates at both Institutions. [unreadable] [unreadable]